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Literature summary for 1.1.1.282 extracted from

  • Peek, J.; Garcia, C.; Lee, J.; Christendat, D.
    Insights into the function of RifI2: structural and biochemical investigation of a new shikimate dehydrogenase family protein (2013), Biochim. Biophys. Acta, 1834, 516-523.
    View publication on PubMed

Cloned(Commentary)

Cloned (Comment) Organism
recombinant expression of His6-tagged wild-type and mutant enzymes in Escherichia coli strain BL21 gold Pseudomonas putida

Crystallization (Commentary)

Crystallization (Comment) Organism
RifI2 X-ray diffraction structure determination and analysis at 2.15 A resolution, modelling Pseudomonas putida

Protein Variants

Protein Variants Comment Organism
N193D site-directed mutagenesis, inactive mutant Pseudomonas putida
N193L site-directed mutagenesis, inactive mutant Pseudomonas putida
N193Q site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Pseudomonas putida
N193S site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Pseudomonas putida

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.43
-
NAD+ pH 8.8, 25┬░C, with quinate Pseudomonas putida
0.71
-
NAD+ pH 8.8, 25┬░C, with shikimate Pseudomonas putida
1.88
-
L-quinate pH 8.8, 25┬░C, with NAD+ Pseudomonas putida
2.18
-
shikimate pH 8.8, 25┬░C, with NADP+ Pseudomonas putida
2.65
-
NADP+ pH 8.8, 25┬░C, with shikimate Pseudomonas putida
3.38
-
shikimate pH 8.8, 25┬░C, with NAD+ Pseudomonas putida

Metals/Ions

Metals/Ions Comment Organism Structure
additional information divalent cations are not required for the activity of RifI2, as the reaction rate is not altered in the presence of 5 mM Ca2+, Mg2+, Mn2+, or Zn2+. Similarly, the reaction is not affected by the addition of 10 mM EDTA to chelate any endogenous divalent cations associated with the enzyme Pseudomonas putida

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
L-quinate + NAD+ Pseudomonas putida
-
3-dehydroquinate + NADH + H+
-
?
L-quinate + NAD+ Pseudomonas putida KT 2240
-
3-dehydroquinate + NADH + H+
-
?
shikimate + NAD+ Pseudomonas putida
-
3-dehydroshikimate + NADH + H+
-
?
shikimate + NAD+ Pseudomonas putida KT 2240
-
3-dehydroshikimate + NADH + H+
-
?

Organism

Organism UniProt Comment Textmining
Pseudomonas putida Q88JP1
-
-
Pseudomonas putida KT 2240 Q88JP1
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant His6-tagged wild-type and mutant enzymes from Escherichia coli strain BL21 gold by nickel affinity chromatography, dialysis, tag cleavage through TEV protease and removal by nickel affinity chromatography, followed by gel filtration Pseudomonas putida

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
L-quinate + NAD+
-
Pseudomonas putida 3-dehydroquinate + NADH + H+
-
?
L-quinate + NAD+
-
Pseudomonas putida KT 2240 3-dehydroquinate + NADH + H+
-
?
shikimate + NAD+
-
Pseudomonas putida 3-dehydroshikimate + NADH + H+
-
?
shikimate + NAD+
-
Pseudomonas putida KT 2240 3-dehydroshikimate + NADH + H+
-
?
shikimate + NADP+ very low activity with NADP+ Pseudomonas putida 3-dehydroshikimate + NADPH + H+
-
?
shikimate + NADP+ very low activity with NADP+ Pseudomonas putida KT 2240 3-dehydroshikimate + NADPH + H+
-
?

Subunits

Subunits Comment Organism
dimer distinct mode of dimerization in which the individual molecules interact in a back-to-front manner, overview. The molecules of the RifI2 dimer associate via hydrophobic interactions between residues on alpha-helix alpha8 of the first molecule and alpha10' of the second molecule. In addition, the side chain of Gln167 on apha-helix alpha8 forms a hydrogen bond with the guanidinium group of Arg243' on beta-strand beta10'. A second hydrogen bond to Arg243' may be made by Asn171. Asp53 and Arg56 on alpha-helix alpha2 bind the imidazole ring of His31' on alpha-helix alpha1' Pseudomonas putida

Synonyms

Synonyms Comment Organism
RifI2
-
Pseudomonas putida
SDH/QDH
-
Pseudomonas putida

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.0055
-
NAD+ pH 8.8, 25┬░C, with quinate Pseudomonas putida
0.0063
-
L-quinate pH 8.8, 25┬░C, with NAD+ Pseudomonas putida
0.022
-
NADP+ pH 8.8, 25┬░C, with shikimate Pseudomonas putida
0.027
-
shikimate pH 8.8, 25┬░C, with NADP+ Pseudomonas putida
2.8
-
NAD+ pH 8.8, 25┬░C, with shikimate Pseudomonas putida
3.9
-
shikimate pH 8.8, 25┬░C, with NAD+ Pseudomonas putida

Cofactor

Cofactor Comment Organism Structure
additional information in the RifI2 structure, the NADP+-specifying motif is replaced with Asp-Pro-Ser-Thr-Ala-Arg (residues 156-161). The side chain of Asp156 binds to the adenine ribose of NAD+, while its negative charge is predicted to repel the additional phosphate of NADP+. RifI2 possesses low apparent binding affinity for NADP+. Analysis of the cofactor-binding sites of the RifI2┬ľNAD+ complex and structure comparison, inportance of the invariant residue Asn193 in the cofactor-binding site, overview Pseudomonas putida
NAD+ the C-terminal domain of each protomer in the RifI2 asymmetric unit contains a bound molecule of NAD+, Cofactor binding structure and mechanism, detailed overview Pseudomonas putida
NADH
-
Pseudomonas putida

General Information

General Information Comment Organism
additional information enzyme RifI2 lacks a conserved C-terminal alpha-helix Pseudomonas putida

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
0.0034
-
L-quinate pH 8.8, 25┬░C, with NAD+ Pseudomonas putida
0.0083
-
NADP+ pH 8.8, 25┬░C, with shikimate Pseudomonas putida
0.012
-
shikimate pH 8.8, 25┬░C, with NADP+ Pseudomonas putida
0.013
-
NAD+ pH 8.8, 25┬░C, with quinate Pseudomonas putida
1.15
-
shikimate pH 8.8, 25┬░C, with NAD+ Pseudomonas putida
3.95
-
NAD+ pH 8.8, 25┬░C, with shikimate Pseudomonas putida